US6057491A - Protein having insecticidal activities and method of use - Google Patents

Protein having insecticidal activities and method of use Download PDF

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US6057491A
US6057491A US09/074,912 US7491298A US6057491A US 6057491 A US6057491 A US 6057491A US 7491298 A US7491298 A US 7491298A US 6057491 A US6057491 A US 6057491A
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sequence
plant
leu
nucleotide sequence
ile
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Amy L. Cigan
Thomas Czapla
Patricia Lynne Fallis
Terry Meyer
Scott A. Mundell
Brian T. Sabus
Karel R. Schubert
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Pioneer Hi Bred International Inc
University of Oklahoma
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Pioneer Hi Bred International Inc
University of Oklahoma
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Application filed by Pioneer Hi Bred International Inc, University of Oklahoma filed Critical Pioneer Hi Bred International Inc
Priority to HU0003403A priority patent/HUP0003403A3/hu
Priority to SI9820048A priority patent/SI20214B/sl
Priority to CZ0421199A priority patent/CZ297319B6/cs
Priority to SK1600-99A priority patent/SK160099A3/sk
Priority to EP98921234A priority patent/EP0986645A1/en
Priority to CA002290776A priority patent/CA2290776C/en
Priority to PCT/US1998/009995 priority patent/WO1998054327A1/en
Priority to AU73891/98A priority patent/AU742709B2/en
Priority to CA002358161A priority patent/CA2358161A1/en
Priority to BR9809516-1A priority patent/BR9809516A/pt
Priority to NZ501218A priority patent/NZ501218A/en
Priority to ARP980102483A priority patent/AR016059A1/es
Assigned to PIONEER HI-BRED INTERNATIONAL, INC. reassignment PIONEER HI-BRED INTERNATIONAL, INC. ASSIGNMENT OF ASSIGNORS INTEREST (SEE DOCUMENT FOR DETAILS). Assignors: FALLIS, PATRICIA LYNNE, CIGAN, AMY L., CZAPLA, TOM, MEYER, TERRY, MUNDELL, SCOTT A., SABUS, BRIAN T.
Assigned to BOARD OF REGENTS FOR UNIVERSITY OF OKLAHOMA reassignment BOARD OF REGENTS FOR UNIVERSITY OF OKLAHOMA ASSIGNMENT OF ASSIGNORS INTEREST (SEE DOCUMENT FOR DETAILS). Assignors: SCHUBERT, KAREL R.
Priority to US09/290,136 priority patent/US6339144B1/en
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
    • C12N15/8241—Phenotypically and genetically modified plants via recombinant DNA technology
    • C12N15/8261—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield
    • C12N15/8271—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield for stress resistance, e.g. heavy metal resistance
    • C12N15/8279—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield for stress resistance, e.g. heavy metal resistance for biotic stress resistance, pathogen resistance, disease resistance
    • C12N15/8286—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield for stress resistance, e.g. heavy metal resistance for biotic stress resistance, pathogen resistance, disease resistance for insect resistance
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
    • A01N65/00—Biocides, pest repellants or attractants, or plant growth regulators containing material from algae, lichens, bryophyta, multi-cellular fungi or plants, or extracts thereof
    • A01N65/08—Magnoliopsida [dicotyledons]
    • A01N65/20—Fabaceae or Leguminosae [Pea or Legume family], e.g. pea, lentil, soybean, clover, acacia, honey locust, derris or millettia
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/415—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from plants
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C12N9/16—Hydrolases (3) acting on ester bonds (3.1)
    • C12N9/18—Carboxylic ester hydrolases (3.1.1)
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
    • Y02A40/00—Adaptation technologies in agriculture, forestry, livestock or agroalimentary production
    • Y02A40/10—Adaptation technologies in agriculture, forestry, livestock or agroalimentary production in agriculture
    • Y02A40/146—Genetically Modified [GMO] plants, e.g. transgenic plants

Definitions

  • the invention relates to compositions and methods for controlling insect species. Additionally, the invention relates to plants and other organisms which have been genetically transformed with the compositions of the invention.
  • Transgenic plants that are resistant to specific insect pests have been produced using genes encoding Bacillus thuringiensis (Bt) endotoxins or plant protease inhibitors (PIs).
  • Bt endotoxin genes have been shown to be effective for control of some insects. Effective plant protection using transgenically inserted PI genetic material has not yet been demonstrated in the field. While cultivars expressing Bt genes may presently exhibit resistance to some insect pests, resistance based on the expression of a single gene might eventually be lost due to the evolution of Bt resistance in the insects. Thus, the search for additional genes which can be inserted into plants to provide protection from insect pests is needed.
  • the corn rootworm (CRW) complex in the United States consists of three species, Diabrotica barberi Smith and Lawrence (Northern), D. undecimpunctata howardi Barber (Southern) and D. virgifera virgifera LeConte (Western).
  • the western and northern species contribute the most to the economic damage to maize.
  • the economic damage and control costs are estimated to exceed one billion dollars a year.
  • Crop rotation is becoming less effective as a CRW control method due to extended diapause in the northern CRW and the development of modified egg laying behavior in western CRW.
  • compositions and methods for the control of insects and other pests are provided.
  • the compositions comprise proteins having pesticidal activities which can be isolated from plants of the genus Pentaclethra. Purified protein, as well as amino acid and DNA sequence information is provided for proteins having rootworm activity.
  • the DNA sequences encoding the pesticidal proteins can be used to transform plants, bacteria, fungi, yeasts, and other organisms for the control of pests.
  • compositions and methods of the invention may be used in a variety of systems for controlling plant and non-plant pests.
  • FIG. 1 provides the amino acid and nucleotide sequence of the cDNA sequence of the corn rootworm active principle, Pentin-1, from Pentaclethra SEQ ID NOS:1 and 2.
  • FIG. 2 provides the amino acid and nucleotide sequence of the CDNA sequence of Pentin-1, optimized for enhanced expression SEQ ID NOS:3 and 4.
  • FIG. 3 provides the amino acid sequence of the Pentin-1 protein with the underlined portion representing the putative signal sequence.
  • the AFS residues immediately following the signal sequence are the first three residues of the mature protein.
  • the ASK residues beginning five residues from the AFS start of the mature protein designates the region of apparent mature amino terminus of pentin-1 expressed as full length protein and proteolyzed in maize roots.
  • FIG. 4 provides the expression cassette for expression of Pentin-1 sequences.
  • compositions and methods for controlling pests are provided.
  • novel pesticidal proteins are provided.
  • the proteins are purified from members of the family Leguminosae, particularly the Leguminous genus Pentaclethra, more particularly the species P. macrophylla and P. macroloba.
  • the pesticidal proteins produced by members of the genus Pentaclethra can be isolated by methods known in the art.
  • Methods for protein isolation include conventional chromatography, including gel-filtration, ion-exchange, and immunoaffinity chromatography, by high-performance liquid chromatography, such as reversed-phase high-performance liquid chromatography, ion-exchange high-performance liquid chromatography, size-exclusion high-performance liquid chromatography, high-performance chromatofocusing and hydrophobic interaction chromatography, etc., by electrophoretic separation, such as one-dimensional gel electrophoresis, two-dimensional gel electrophoresis, etc. See for example Current Protocols in Molecular Biology, Vols. 1 and 2, Ausubel et al. (eds.), John Wiley & Sons, NY (1988), herein incorporated by reference.
  • the protein, or the polypeptides of which it is comprised can be characterized and sequenced by standard methods known in the art.
  • the purified protein, or the polypeptides of which it is comprised may be fragmented as with cyanogen bromide, or with proteases such as papain, chymotrypsin, trypsin, lysyl-C endopeptidase, etc. (Oike et al. (1982) J. Biol. Chem. 257:9751-9758; Liu et al. (1983) Int. J. Pept. Protein Res. 21:209-215).
  • the resulting peptides are separated, preferably by HPLC, or by resolution of gels and electroblotting onto PVDF membranes, and subjected to amino acid sequencing. To accomplish this task, the peptides are preferably analyzed by automated sequenators. It is recognized that N-terminal, C-terminal, or internal amino acid sequences can be determined. From the amino acid sequence of the purified protein, a nucleotide sequence can be synthesized which can be used as a probe to aid in the isolation of the gene encoding the pesticidal protein.
  • antibodies raised against partially purified or purified peptides can be used to determine the spatial and temporal distribution of the protein of interest.
  • tissue where the protein is most abundant, and possibly more highly expressed can be determined and expression libraries constructed.
  • Methods for antibody production are known in the art. See, for example Antibodies, A Laboratory Manual, Harlow and Lane (eds.), Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. (1988), and the references cited therein. See also, Radka et al. (1983) J. Immunol. 128:2804; and Radka et al. (1984) Immunogenetics 19:63.
  • Such antibodies can be used to isolate proteins with similar binding domains and the proteins tested for activity against insect pests of interest.
  • any combination of methods may be utilized to purify proteins having pesticidal properties. As an isolation protocol is being determined, the pesticidal activity can be tested for each fraction of material obtained after each purification step.
  • substantially purified protein fraction By “substantially purified” or “substantially pure” is intended protein which is substantially free of any compound normally associated with the protein in its natural state. “Substantially pure” preparations of protein can be assessed by the absence of other detectable protein bands following SDS-PAGE as determined visually or by densitometry scanning. Alternatively, the absence of other amino-terminal sequences or N-terminal residues in a purified preparation can indicate the level of purity. Purity can be verified by rechromatography of "pure” preparations showing the absence of other peaks by ion exchange, reverse phase or capillary electrophoresis.
  • substantially pure or “substantially purified” are not meant to exclude artificial or synthetic mixtures of the proteins with other compounds.
  • the terms are also not meant to exclude the presence of minor impurities which do not interfere with the biological activity of the protein, and which may be present, for example, due to incomplete purification.
  • the entire nucleotide sequence encoding the protein can be determined by PCR experiments. Likewise, fragments obtained from PCR experiments can be used to isolate cDNA sequences from expression libraries. See, for example, Molecular Cloning, A Laboratory Manual, Second Edition, Vols. 1-3, Sambrook el al. (eds.) Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. (1989), and the references cited therein.
  • proteins and the nucleotide sequences encoding such proteins can be isolated which are inhibitory or toxic to particular insect species.
  • Such proteins and nucleotide sequences of the invention can be utilized to protect plants from pests, including insects, fungi, bacteria, nematodes, viruses or viroids, and the like, particularly insect pests.
  • proteins and nucleotide sequences which are inhibitory or toxic to insects of the order Coleoptera can be obtained.
  • Insect pests include insects selected from the orders Coleoptera, Diptera, Hymenoptera, Lepidoptera, Mallophaga, Homoptera, Hemiptera, Orthroptera, Thysanoptera, Dermaptera, Isoptera, Anoplura, Siphonaptera, Trichoptera, etc., particularly Coleoptera.
  • Insect pests of the invention for the major crops include:
  • nucleotide sequences of the invention can be used to isolate other homologous sequences in other plant species, particularly other Leguminous species. Methods are readily available in the art for the hybridization of nucleic acid sequences. Coding sequences from other plants may be isolated according to well known techniques based on their sequence homology to the coding sequences set forth herein. In these techniques all or part of the known coding sequence is used as a probe which selectively hybridizes to other pesticidal coding sequences present in a population of cloned genomic DNA fragments or cDNA fragments (i.e. genomic or cDNA libraries) from a chosen organism.
  • the entire Pentin-1 sequence or portions thereof may be used as probes capable of specifically hybridizing to corresponding coding sequences and messenger RNAs.
  • probes include sequences that are unique and are preferably at least about 10 nucleotides in length, and most preferably at least about 20 nucleotides in length.
  • Such probes may be used to amplify Pentin-1 coding sequences from a chosen organism by the well-known process of polymerase chain reaction (PCR). This technique may be used to isolate additional coding sequences from a desired organism or as a diagnostic assay to determine the presence of Pentin-1 coding sequences in an organism.
  • PCR polymerase chain reaction
  • Such techniques include hybridization screening of plated DNA libraries (either plaques or colonies; see, e.g. Sambrook et al., Molecular Cloning, eds., Cold Spring Harbor Laboratory Press (1989)) and amplification by PCR using oligonucleotide primers corresponding to sequence domains conserved among the amino acid sequences (see, e.g. Innis et al., PCR Protocols, a Guide to Methods and Applications, eds., Academic Press (1990)).
  • hybridization of such sequences may be carried out under conditions of reduced stringency, medium stringency or even stringent conditions (e.g., conditions represented by a wash stringency of 35-40% formamide with 5 ⁇ Denhardt's solution, 0.5% SDS and 1 ⁇ SSPE at 37° C.; conditions represented by a wash stringency of 40-45% formamide with 5 ⁇ Denhardt's solution, 0.5% SDS, and 1 ⁇ SSPE at 42° C.; and conditions represented by a wash stringency of 50% formamide with 5 ⁇ Denhardt's solution, 0.5% SDS and 1 ⁇ SSPE at 42° C., respectively), to DNA encoding the insecticidal genes disclosed herein in a standard hybridization assay. See J. Sambrook et al., Molecular Cloning, A Laboratory Manual 2d Ed. (1989) Cold Spring Harbor Laboratory.
  • stringent conditions or “stringent hybridization conditions” includes reference to conditions under which a probe will hybridize to its target sequence, to a detectably greater degree than other sequences (e.g., at least 2-fold over background). Stringent conditions are sequence-dependent and will be different in different circumstances. By controlling the stringency of the hybridization and/or washing conditions, target sequences can be identified which are 100% complementary to the probe (homologous probing). Alternatively, stringency conditions can be adjusted to allow some mismatching in sequences so that lower degrees of similarity are detected (heterologous probing). Generally, a probe is less than about 1,000 nucleotides in length, preferably less than about 500 nucleotides in length, typically from about 50 to about 300 nucleotides in length.
  • stringent conditions will be those in which the salt concentration is less than about 1.5 M Na ion, typically about 0.01 to 1.0 M Na ion concentration (or other salts) at pH 7.0 to 8.3 and the temperature is at least about 30 for short probes (e.g., 10 to 50 nucleotides) and at least about 60° C. for long probes (e.g., greater than 50 nucleotides).
  • Stringent conditions may also be achieved with the addition of destabilizing agents such as formamide.
  • Exemplary moderate stringency conditions include hybridization in 40 to 45% formamide, 1 M NaCl, 1% SDS at 37° C., and a wash in 0.5 ⁇ to 1 ⁇ SSC at 55° C to 60° C.
  • Exemplary high stringency conditions include hybridization in 50% formamide, 1 M NaCl, 1% SDS at 37° C., and a wash in 0.1 ⁇ SSC at 60° C. to 65° C.
  • Tm can be approximated from the equation of Meinkoth and Wahl, Anal. Biochem.
  • Tm 81.5C+16.6 (log M)+0.41 (%GC)-0.61 (% form)-500/L; where M is the molarity of monovalent cations, %GC is the percentage of guanosine and cytosine nucleotides in the DNA, % form is the percentage of formamide in guanosine and cytosine nucleotides in the DNA, % form is the percentage of formamide in the hybridization solution, and L is the length of the hybrid in base pairs.
  • the Tm is the temperature (under defined ionic strength and pH) at which 50% of a complementary target sequence hybridizes to a perfectly matched probe. Tm is reduced by about 1° C.
  • Tm, hybridization and/or wash conditions can be adjusted to hybridize to sequences of the desired identity. For example, if sequences with ⁇ 90% identity are sought, the Tm can be decreased 10° C.
  • stringent conditions are selected to be about 5° C. lower than the thermal melting point (Tm) for the specific sequence and its complement at a defined ionic strength and pH.
  • Tm thermal melting point
  • severely stringent conditions can utilize a hybridization and/or wash at 1, 2, 3, or 4° C. lower than the thermal melting point (Tm); moderately stringent conditions can utilize a hybridization and/or wash at 6, 7, 8 or 9 or 10° C.
  • Tm thermal melting point
  • low stringency conditions can utilize a hybridization and/or wash at 11, 12, 13, 14, 15, or 20° C. lower than the thermal melting point (Tm)
  • Tm thermal melting point
  • sequences which code for Pentin-1 and other insecticidal proteins of the invention and hybridize to the gene disclosed herein will be at least about 50% homologous, about 70% homologous, up to about 85% homologous or more up to about 90% to about 95% homologous with the disclosed sequence. That is, the sequence similarity of sequences may range, sharing at least about 50%, about 70%, and about 85% up to about 90% to 95% sequence similarity.
  • sequence relationships between two or more nucleic acids or polynucleotides are used to describe the sequence relationships between two or more nucleic acids or polynucleotides: (a) “reference sequence”, (b) “comparison window”, (c) “sequence identity”, (d) “percentage of sequence identity”, and (e) “substantial identity”.
  • reference sequence is a defined sequence used as a basis for sequence comparison.
  • a reference sequence may be a subset or the entirety of a specified sequence; for example, as a segment of a full-length cDNA or gene sequence, or the complete cDNA or gene sequence.
  • comparison window means includes reference to a contiguous and specified segment of a polynucleotide sequence, wherein the polynucleotide sequence may be compared to a reference sequence and wherein the portion of the polynucleotide sequence in the comparison window may comprise additions or deletions (i.e., gaps) compared to the reference sequence (which does not comprise additions or deletions) for optimal alignment of the two sequences.
  • the comparison window is at least 20 contiguous nucleotides in length, and optionally can be 30, 40, 50, 100 or longer.
  • Optimal alignment of sequences for comparison may be conducted by the local homology algorithm of Smith and Waterman, Adv. Appl. Math. 2:482 . (1981); by the homology alignment algorithm of Needleman and Wunsch, J. Mol. Biol. 48:443 (1970); by the search for similarity method of Pearson and Lipman, Proc. Natl. Acad. Sci.
  • the BLAST family of programs which can be used for database similarity searches includes: BLASTN for nucleotide query sequences against nucleotide sequences; BLASTX for nucleotide query sequences against protein database sequences; BLASTP for protein query sequences against protein database sequences; TBLASTN for protein query sequences against nucleotide database sequences; and TBLASTX for nucleotide query sequences against nucleotide database sequences.
  • BLAST searches assume that proteins can be modeled as random sequences. However, many real proteins comprise regions of nonrandom sequences which may be homopolymeric tracts, short-period repeats, or regions enriched in one or more amino acids. Such low-complexity regions may be aligned between unrelated proteins even though other regions of the protein are entirely dissimilar.
  • a number of low-complexity filter programs can be employed to reduce such low-complexity alignments. For example, the SEG (Wooten and Federhen, Comput. Chem. 17:149-163 (1993) and XNU (Claverie and States, Comput. Chem., 17:191-201 (1993)) low-complexity filters can be employed alone or in combination.
  • sequence identity in the context of two nucleic acid or polypeptide sequences includes reference to the residues in the two sequences which are the same when aligned for maximum correspondence over a specified comparison window.
  • sequence identity When percentage of sequence identity is used in reference to proteins it is recognized that residue positions which are not identical often differ by conservative amino acid substitutions, where amino acid residues are substituted for other amino acid residues with similar chemical properties (e.g. charge or hydrophobicity) and therefore do not change the functional properties of the molecule. Where sequences differ in conservative substitutions, the percent sequence identity may be adjusted upwards to correct for the conservative nature of the substitution. Sequences which differ by such conservative substitutions are said to have "sequence similarity" or “similarity”.
  • Means for making this adjustment are well-known to those of skill in the art. Typically this involves scoring a conservative substitution as a partial rather than a full mismatch, thereby increasing the percentage sequence identity. Thus, for example, where an identical amino acid is given a score of 1 and a non-conservative substitution is given a score of zero, a conservative substitution is given a score between zero and 1.
  • the scoring of conservative substitutions is calculated, e.g., according to the algorithm of Meyers and Miller, Computer Applic. Biol. Sci. 4:11-17 (1988) e.g., as implemented in the program PC/GENE (Intelligenetics, Mountain View, Calif., USA).
  • percentage of sequence identity means the value determined by comparing two optimally aligned sequences over a comparison window, wherein the portion of the polynucleotide sequence in the comparison window may comprise additions or deletions (i.e., gaps) as compared to the reference sequence (which does not comprise additions or deletions) for optimal alignment of the two sequences. The percentage is calculated by determining the number of positions at which the identical nucleic acid base or amino acid residue occurs in both sequences to yield the number of matched positions, dividing the number of matched positions by the total number of positions in the window of comparison and multiplying the result by 100 to yield the percentage of sequence identity.
  • polynucleotide sequences means that a polynucleotide comprises a sequence that has at least 70% sequence identity, preferably at least 80%, more preferably at least 90% and most preferably at least 95%, compared to a reference sequence using one of the alignment programs described using standard parameters.
  • sequence identity preferably at least 80%, more preferably at least 90% and most preferably at least 95%.
  • nucleotide sequences are substantially identical as if two molecules hybridize to each other under stringent conditions. However, nucleic acids which do not hybridize to each other under stringent conditions are still substantially identical if the polypeptides which they encode are substantially identical. This may occur, e.g., when a copy of a nucleic acid is created using the maximum codon degeneracy permitted by the genetic code.
  • One indication that two nucleic acid sequences are substantially identical is that the polypeptide which the first nucleic acid encodes is immunologically cross reactive with the polypeptide encoded by the second nucleic acid.
  • substantially identical in the context of a peptide indicates that a peptide comprises a sequence with at least 70% sequence identity to a reference sequence, preferably 80%, more preferably 85%, most preferably at least 90% or 95% sequence identity to the reference sequence over a specified comparison window.
  • optimal alignment is conducted using the homology alignment algorithm of Needleman and Wunsch, J. Mol. Biol. 48:443 (1970).
  • An indication that two peptide sequences are substantially identical is that one peptide is immunologically reactive with antibodies raised against the second peptide.
  • a peptide is substantially identical to a second peptide, for example, where the two peptides differ only by a conservative substitution.
  • Peptides which are "substantially similar” share sequences as noted above except that residue positions which are not identical may differ by conservative amino acid changes.
  • the pesticidal proteins may be oligomeric and will vary in molecular weight, number of promoters, component peptides, activity against particular pests, and in other characteristics.
  • proteins active against a variety of pests may be isolated and characterized.
  • the purified or partially purified proteins of the invention are tested for insecticidal activity against corn rootworm, including Diabrotica barberi (Northern), D. undecimpunctata howardi (Southern), and D. virgifera vergifera (Western).
  • Pentin-1 is a glycosylated protein of approximately 45 to about 50 kdal.
  • the amino acid and nucleotide sequence of the Pentin-1 protein is given in FIG. 1 and SEQ ID NOS: 1 and 2.
  • Pentin-1 The highest concentration of Pentin-1 in the plant appears to be in mature seeds.
  • the protein is heat stable and has an LC50 of approximately 10 ⁇ g/ml of diet against corn rootworm.
  • Pentin-1 and other proteins of the invention may be altered in various ways including amino acid substitutions, deletions, truncations, and insertions. Methods for such manipulations are generally known in the art.
  • amino acid sequence variants of the pesticidal proteins can be prepared by mutations in the DNA. Methods for mutagenesis and nucleotide sequence alterations are well known in the art. See, for example, Kunkel, T. (1985) Proc. Natl. Acad. Sci. USA 82:488-492; Kunkel et al. (1987) Methods in Enzymol. 154:367-382; U.S. Pat. No.
  • genes and nucleotide sequences of the invention include both the naturally occurring sequences as well as mutant forms.
  • proteins of the invention encompass both naturally occurring proteins as well as variations and modified forms thereof. Such variants will continue to possess the desired pesticidal activity.
  • mutations that will be made in the DNA encoding the variant must not place the sequence out of reading frame and preferably will not create complementary regions that could produce secondary mRNA structure. See, EP Patent Application Publication No. 75,444.
  • the present invention encompasses the pesticidal proteins as well as components and fragments thereof. That is, it is recognized that component promoters, polypeptides or fragments of the proteins may be produced which retain pesticidal activity. These fragments include truncated sequences, as well as N-terminal, C-terminal, internal and internally deleted amino acid sequences of the proteins.
  • deletions, insertions, and substitutions of the protein sequence are not expected to produce radical changes in the characteristics of the protein.
  • the effect will be evaluated by routine screening assays. That is, the activity can be evaluated by insect toxicity assay.
  • DNA shuffling is a process for recursive recombination and mutation, performed by random fragmentation of a pool of related genes, followed by reassembly of the fragments by primerless PCR.
  • DNA shuffling is a process for recursive recombination and mutation, performed by random fragmentation of a pool of related genes, followed by reassembly of the fragments by primerless PCR.
  • DNA shuffling of a rational design is that shuffling can optimize the function of genes without first determining which gene product is rate limiting.
  • the present invention provides methods for sequenced shuffling utilizing polypeptides of the invention, and compositions resulting therefrom.
  • sequenced shuffling provides a means for generating libraries of polynucleotides having a desired characteristic which can be selected or screened for.
  • Libraries of recombinant polypeptides are generated from a population of related sequence polypeptides that comprise sequenced regions which have substantial sequence identity and can be homologously recombined in vitro or in vivo.
  • the population of sequenced-recombined polynucleotides comprises a subpopulation of polynucleotides which possess desired or advantageous characteristics and can be selected by a suitable selection or screening method.
  • the characteristics can be any property or attribute capable of being selected for or detected in a screening system, and may include properties of: an encoded protein, a transcriptional element, a sequence controlling transcription, RNA processing, RNA stability, chromatin confirmation, translation, or other expression property of a gene or transgene, a replicative element, a protein-binding element, or the like, such as any feature which confers a selectable or detectable property.
  • the selected characteristic will be an increased Km and/or Kcat over the wild-type protein as provided herein.
  • a protein or polynucleotide generated from sequenced shuffling will have a ligand binding affinity greater than the non-shuffled wild-type polynucleotide.
  • the increase in such properties can be at least 110%, 120%, 130%, 140% or at least 150% of the wild-type value.
  • Pentin-1 is a member of a broader gene family of esterases, and more specifically lipid acyl hydrolases as determined by sequence similarity. Gene shuffling is a method that can improve or alter a biological activity of a given gene product. Gene shuffling, in conjunction with a selection strategy, can be used to improve properties such as substrate specificity, solubility, temperature and pH optima of a protein or enzyme by directed molecular evolution. In the case of Pentin-1 toxicity toward insects as determined by the lethal concentrations is a most relevant parameter.
  • Gene shuffling can be applied to a single gene which introduces mutations within that gene at a given frequency. Combinations of synergistic mutations can then be selected by subsequent generations of gene shuffling from the primary mutant population. This approach can be applied to Pentin-1.
  • different members of gene families that are already encoded by divergent but related sequences can be used for gene shuffling. These could include but not be limited to Pentin-1, from Pentaclethra and an expressed sequence tag from maize identified as 5C9 that encodes a cDNA that is about 57% identical to Pentin-1 at the nucleotide level. See copending patent application Ser. No. 08/449,986 filed May 25, 1995, herein incorporated by reference. Concomitantly mutations will also be introduced by gene shuffling further contributing to the genetic diversity. Then synergistic combinations of fusions between the members of the gene family and newly introduced mutations can be selected by directed molecular evolution strategies.
  • Lipid acyl hydrolases comprise a diverse multigene family that is conserved across many plant species.
  • the enzymes exhibit hydrolyzing activity for many glyco- and phospholipids.
  • Substrates include monogalactosyldiacylglycerol, acylsterylgucoside, phosphatidylcholine, lysophosphatidylcholine, phosphatidylethanolamine, lysophosphatidylethanolamine, phosphatidylinositol, as well as many other lipid substrates.
  • membrane composition of various insects as well as plants can vary from species to species and can be affected by diet or growth conditions. Consequently, the activity of a given lipid acyl hydrolase for a given substrate could affect both specificity and potency. Altered substrate specificity could be one parameter for selection of products of gene shuffling.
  • Solubility and protein stability could also be selected from shuffled gene products.
  • Insecticidal proteins are active in the harsh environment of the insect gut lumen. Their proteins are digested by proteases, and affected by reducing or oxidizing conditions that vary according to the insect species tested.
  • the solubility and stability of lipid acyl hydrolases both in the transgenic plant and in the insect gut lumen could affect biological activity and could be altered through gene shuffling strategies.
  • Conditions for the enzyme reaction such as pH and temperature optima may also affect the insecticidal activity of the Pentin-1.
  • the gut pH of corn rootworm is 5.5-6.0.
  • Selection of shuffled Pentin-1 gene products for enzymatic activity toward lipid substrates in this pH range is another parameter that could affect toxicity.
  • the pentin sequence of the present invention can be utilized in gene shuffling experiments with other lipid hydrolases such as patatins, and in particular with 5C9.
  • insecticidal proteins include those from Bacillus, including ⁇ -endotoxins and vegetative insecticidal proteins, as well as protease inhibitors (both serine and cysteine types), lectins, cc-amylases, peroxidases, cholesterol oxidase, and the like.
  • expression of the proteins of the invention in a transgenic plant is accompanied by the expression of one or more Bacillus thuringiensis (Bt) ⁇ -endotoxins.
  • Bt Bacillus thuringiensis
  • This co-expression of more than one insecticidal principle in the same transgenic plant can be achieved by genetically engineering a plant to contain and express all the genes necessary.
  • a plant, Parent 1 can be genetically engineered for the expression of proteins of the invention.
  • a second plant, Parent 2 can be genetically engineered for the expression of other principles, such as a Bt ⁇ -endotoxin.
  • the present invention also encompasses nucleotide sequences from organisms other than Pentaclethra, where the proteins cross-react with antibodies raised against the proteins of the invention or where the nucleotide sequences are isolatable by hybridization with the nucleotide sequences of the invention.
  • the proteins isolated or those encoded by such nucleotide sequences can be tested for pesticidal activity.
  • the isolated proteins can be assayed for pesticidal activity by the methods disclosed herein or others well-known in the art.
  • the proteins of the invention can be used in combination with seed coatings available in the art.
  • transformed seed are coated with applications of available insecticide sprays or powders.
  • insecticides are known in the art. See, for example, U.S. Pat. Nos. 5,696,144; 5,695,763; 5,420,318; 5,405,612; 4,596,206; 4,356,934; 4,886,541; etc., herein incorporated by reference.
  • nucleotide sequences encoding the pesticidal proteins of the invention can be manipulated and used to express the protein in a variety of hosts including other organisms, including microorganisms and plants.
  • the proteins of the invention may be used for protecting agricultural crops and products from pests by introduction via a suitable vector into a microbial host, and said host applied to the environment or plants.
  • Microorganism hosts may be selected which are known to occupy the "phytosphere" (phylloplane, phyllosphere, rhizosphere, and/or rhizoplana) of one or more crops of interest. These microorganisms are selected so as to be capable of successfully competing in the particular environment with the wild-type microorganisms, provide for stable maintenance and expression of the gene expressing the polypeptide pesticide, and, desirably, provide for improved protection of the pesticide from environmental degradation and inactivation.
  • the proteins of the invention can be used in expression cassettes for expression in any host of interest.
  • Such expression cassettes will comprise a transcriptional initiation region linked to the gene encoding the pesticidal gene of interest.
  • Such an expression cassette is provided with a plurality of restriction sites for insertion of the gene of interest to be under the transcriptional regulation of the regulatory regions.
  • the expression cassette may additionally contain selectable marker genes suitable for the particular host organism.
  • the transcriptional initiation region may be native or analogous or foreign or heterologous to the host. Additionally, the promoter may be the natural sequence or alternatively a synthetic sequence. By foreign is intended that the transcriptional initiation region is not found in the wild-type host into which the transcriptional initiation region is introduced.
  • a chimeric gene comprises a coding sequence operably linked to transcription initiation region which is heterologous to the coding sequence. While any promoter or promoter element capable of driving expression of a coding sequence can be utilized, of particular interest for expression in plants are root promoters (Bevan et al. (1993) in Gene Conservation and Exploitation. Proceedings of The 20th Stadler Genetics Symposium, Gustafson et al.
  • the transcriptional cassette will include in 5'-3' direction of transcription, a transcriptional and translational initiation region, a DNA sequence of interest, and a transcriptional and translational termination region functional in plants.
  • the termination region may be native with the transcriptional initiation region, may be native with the DNA sequence of interest, or may be derived from another source.
  • Convenient termination regions are available from the Ti-plasmid of A. tumefaciens, such as the octopine synthase and nopaline synthase termination regions. See also, Guerineau et al., (1991) Mol. Gen. Genet. 262:141-144; Proudfoot (1991) Cell 64:671-674; Sanfacon et al. (1991) Genes Dev.
  • the nucleotide sequences encoding the proteins or polypeptides of the invention are particularly useful in the genetic manipulation of plants.
  • the genes of the invention are provided in expression cassettes for expression in the plant of interest.
  • the cassette will include 5' and 3' regulatory sequences operably linked to the gene of interest.
  • the cassette may additionally contain at least one additional gene to be cotransformed or linked and transformed into the organism.
  • the gene(s) of interest can be provided on another expression cassette.
  • the gene(s) may be optimized for increased expression in the transformed plant. That is, the genes can be synthesized using plant preferred codons for improved expression. Methods are available in the art for synthesizing plant preferred genes. See, for example, U.S. Pat. Nos. 5,380,831, 5,436,391, and Murray et al. (1989) Nucleic Acids Res. 17:477-498, herein incorporated by reference.
  • nucleotide sequences can be optimized for expression in any plant. It is recognized that all or any part of the gene sequence may be optimized or synthetic. That is, synthetic or partially optimized sequences may also be used.
  • Additional sequence modifications are known to enhance gene expression in a cellular host. These include elimination of sequences encoding spurious polyadenylation signals, exon-intron splice site signals, transposon-like repeats, and other such well-characterized sequences which may be deleterious to gene expression.
  • the G-C content of the sequence may be adjusted to levels average for a given cellular host, as calculated by reference to known genes expressed in the host cell. When possible, the sequence may be modified to avoid predicted hairpin secondary mRNA structures.
  • the expression cassettes may additionally contain 5' leader sequences in the expression cassette construct.
  • leader sequences can act to enhance translation.
  • Translation leaders are known in the art and include: picornavirus leaders, for example, EMCV leader (Encephalomyocarditis 5' noncoding region) (Elroy-Stein, O., Fuerst, T. R., and Moss, B. (1989) PNAS USA, 86:6126-6130); potyvirus leaders, for example, TEV leader (Tobacco Etch Virus) (Allison et al. (1986)); MDMV leader (Maize Dwarf Mosaic Virus) Virology, 154:9-20); and human immunoglobulin heavy-chain binding protein (BiP), (Macejak, D.
  • EMCV leader Engelphalomyocarditis 5' noncoding region
  • potyvirus leaders for example, TEV leader (Tobacco Etch Virus) (Allison et al. (1986)
  • MDMV leader Maize Dwarf
  • the genes of the present invention can be targeted to the chloroplast or amyloplast for expression.
  • the expression cassette will additionally contain a gene encoding a transit peptide to direct the gene of interest to the chloroplasts.
  • transit peptides are known in the art. See, for example, Von Heijne et al. (1991) Plant Mol. Biol. Rep. 9:104-126; Clark et al. (1989) J. Biol. Chem. 264:17544-17550; della-Cioppa et al. (1987) Plant Physiol. 84:965-968; Romer et al. (1993) Biochem. Biophys. Res Commun. 196:1414-1421; and Shah et al. (1986) Science 233:478-481.
  • the construct may also include any other necessary regulators such as nuclear localization signals (Kalderon et al. (1984) Cell 39:499-509; and Lassner et al. (1991) Plant Molecular Biology 17:229-234); plant translational consensus sequences (Joshi, C. P. (1987) Nucleic Acids Research 15:6643-6653), introns (Luehrsen and Walbot (1991) Mol. Gen. Genet. 225:81-93) and the like, operably linked to the nucleotide sequence of interest.
  • the protein can be expressed comprising the native signal sequence. See FIG. 3.
  • other signal sequences in the art for example the barley alpha amylase signal sequence, may be utilized.
  • the various DNA fragments may be manipulated, so as to provide for the DNA sequences in the proper orientation and, as appropriate, in the proper reading frame.
  • adapters or linkers may be employed to join the DNA fragments or other manipulations may be involved to provide for convenient restriction sites, removal of superfluous DNA, removal of restriction sites, or the like.
  • in vitro mutagenesis, primer repair, restriction, annealing, resection, ligation, PCR, or the like may be employed, where insertions, deletions or substitutions, e.g. transitions and transversions, may be involved.
  • compositions of the present invention can be used to transform any plant.
  • Genetically modified plants, plant cells, plant tissue, seed, and the like can be obtained. Transformation protocols may vary depending on the type of plant or plant cell, i.e. monocot or dicot, targeted for transformation. Suitable methods of transforming plant cells include microinjection (Crossway et al. (1986) Biotechniques 4:320-334), electroporation (Riggs et al. (1986) Proc. Natl. Acad. Sci. USA, 83:5602-5606, Agrobacterium mediated transformation (Hinchee et al. (1988) Biotechnology, 6:915-921), direct gene transfer (Paszkowski et al.
  • the plant plastid can be transformed directly.
  • Stable transformation of plastids have been reported in higher plants, see, for example, SVAB et al. (1990) Proc. Natl. Acad. Sci. USA 87:8526-8530; SVAB & Maliga (1993) Proc. Natl. Acad. Sci. USA 90:913-917; Staub & Maliga (1993) Embo J. 12:601-606.
  • the method relies on particle gun delivery of DNA containing a selectable marker and targeting of the DNA to the plastid genome through homologous recombination.
  • plastid transformation can be accomplished by transactivation of a silent plastid-borne transgene by tissue-specific expression of a nuclear-encoded and plastid-directed RNA polymerase.
  • tissue-specific expression of a nuclear-encoded and plastid-directed RNA polymerase has been reported in McBride et al. (1994) Proc. Natl. Acad. Sci. USA 91:7301-7305.
  • the cells which have been transformed may be grown into plants in accordance with conventional ways. See, for example, McCormick et al. (1986) Plant Cell Reports, 5:81-84. These plants may then be grown, and either pollinated with the same transformed strain or different strains, and the resulting offspring having the desired phenotypic characteristic identified. Two or more generations may be grown to ensure that the subject phenotypic characteristic is stably maintained and inherited and then seeds harvested to ensure the desired phenotype or other property has been achieved.
  • the proteins will be expressed in the transformed organisms in amounts to be toxic to the insects of interest or inhibitory to insect growth.
  • P. macroloba seeds were collected from the lowland moist forest of Costa Rica and transported to the inventors' laboratories where they were sliced, lyophilized and stored at -20° C. prior to use. Frozen seeds were diced into smaller pieces and homogenized using a Brinkmann homogenizer. In a typical procedure, 10 grams of seed material was homogenized with 1-2 grams of insoluble polyvinylpyrrolidone and 50-100 ml of 10 mM sodium phosphate buffer, pH 7.5. The homogenate was then stirred at 4° C. for 8-10 hours and centrifuged at 5,000 rpm for 15 minutes.
  • the supernatant fluid was carefully decanted and poured through a single layer of Miracloth, and collected so as to avoid the transfer of lipid-like materials in the extract which have separated and solidified on the surface during centrifugation.
  • the pellet was discarded and the collected liquid, which was still somewhat cloudy, was centrifuged a second time at 18,000 rpm in a Sorvall SS-34 rotor, or its equivalent, for 30 minutes.
  • the crude and crude dialyzed extracts were analyzed for protein composition or content, and were found to contain a substance which was an insecticidally active against corn rootworm (CRW) in biological assays.
  • the insecticide was found to be a protein or proteinaceous substance.
  • a 100-ml sample of the dialyzed crude extract was heated to about 80° C. using a water bath and held at this temperature for about 5 minutes.
  • the heated extract was then cooled below 25° C. using an ice bath and, after cooling, centrifuged for 15-30 minutes at 18,000 rpm using a Sorval SS-34 rotor.
  • the clear supernatant liquid was removed, saved and designated hereafter as heat-treated extract.
  • the pelleted material was discarded. It was noted that the heat-treated extract sometimes exhibited a tendency to gel.
  • the heat-treated extract was assayed protein for using the Bradford method with BSA as the standard and was found to possess insecticidal activity against CRW in biological assays.
  • a sample of the heated extract was fractionated and concentrated using ammonium sulfate.
  • the sample was cooled using an ice bath and powdered ammonium sulfate, 0.6 g/ml sample, was slowly added with stirring. Once the ammonium sulfate addition was completed, the sample was maintained at ice bath temperatures for about 30 minutes.
  • the sample was then centrifuged at 4° C. for 20 minutes at 18,000 rpm using a Sorval SS-34 rotor. The supernatant liquid and the pelleted material were separated, and the pelleted material was resolubilized in a minimum amount of 10 mM sodium phosphate buffer, pH 7.5, and dialyzed extensively against 10 mM sodium phosphate buffer, pH 7.5.
  • the supernatant liquid and the resolubilized pelleted material were assayed for protein content by the Bradford method using BSA as the standard and tested for biological activity against CRW. The majority of the Pentin-1 was found in the pelleted material and it was insecticidal against CRW. Alternatively, the volume of the heated extract was reduced by centrifugal concentration using CentriconTM or similar concentrating devices according to the manufacturer's directions.
  • the proteins were also fractionated by size-exclusion chromatography on either a Pharmacia Sephacryl S-200 column or a Pharmacia Superose 12 column.
  • the column were equilibrated with at least two to three column volumes of 10 mM sodium phosphate buffer, pH 7.5, before the sample was applied to the column.
  • the proteins were eluted from the column with 10 mM sodium phosphate buffer, pH 7.5.
  • the fractions were assayed for protein content by the Bradford method using BSA as the standard and were bioassayed using corn rootworm larvae.
  • Heat-treated samples or samples which had been subjected to size-exclusion chromatography were fractionated by anion exchange chromatography using either a Pharmacia Q Sepharose column or a Pharmacia Resource Q column. Prior to placement of the sample on the column, the column was first washed with 25 mM Tris-HCl or suitable buffer containing 1 M NaCl, and then equilibrated with the same buffer without NaCl.
  • the pH of the buffers used in the chromatography ranged between pH 4 and pH 10.
  • chromatography using 25 mM Tris-HCl buffer, pH 9.0 is described herein.
  • the sample Prior to injecting the sample onto the column, the sample was dialyzed using a 3,500 MWCO membrane through 2-3 exchanges of 25 mM Tris-HCl buffer without 1 M NaCl. After placement on the column, the flow-through was collected and the column was washed with 25 mM Tris-HCl, pH 9.0. The wash was also collected. The column was then eluted with a gradient ranging from 25 mM Tris-HCl, pH 9.0, no NaCl to 25 mM Tris-HCl, pH 9.0, 1 M NaCl. All fractions collected were dialyzed with a minimum of two buffer exchanges against 10 mM sodium phosphate, pH 7.5.
  • the flow-through, wash and the salt-eluted fractions were assayed for protein by the Bradford method using BSA as the standard and bioassayed using CRW. Active material was found in the flow-through and in fractions which were eluted between 0.2 and 0.5 M NaCl. To determine whether the capacity of the column was exceeded, resulting in additional materials passing through the column without binding. the active material in the flow-through was reapplied to the column after re-equilibration. Most of the UV 280 nm absorbing material passed through the column. These observations suggested this active material has different properties than the material which bound to the column and was eluted with the increasing increments of NaCl.
  • the other buffers used were also suitable for anion exchange chromatography as known to those familiar with the art. Active material could also be purified by cation-exchange chromatography.
  • Pentin-1 material was purified to near homogeneity by size-exclusion chromatography or anion-exchange chromatography. Minor protein bands were removed by high pressure liquid chromatography (HPLC) using a reverse phase column prior to amino acid analysis and determination of the amino acid sequence.
  • HPLC high pressure liquid chromatography
  • the purity of the samples and the subunit molecular weight were determined by SDS-PAGE using 12% polyacrylamide gels and generally following the method of Laemmli, Nature 227:680-685 (1970). Gels were stained with either Coomassie Blue R250 using standard protocols or silver stained (Hammer et al., Phytochemistry 28:3019-3026 (1989)). By SDS-PAGE, the subunit molecular weight of the active substance was found to be in the range 40-55,000 ⁇ 5,000 Daltons.
  • an extract can be subjected to isoelectric focusing (IEF) using the Rotofor system (Bio-Rad).
  • the Rotofor separates molecules on the basis of their pI or isoelectric point. Every molecule will have a specific charge, either positive or negative, at a specific pH.
  • the Rotofor using an electrical current, moves molecules through a pH gradient until they reach their pI; i.e., the pH at which they have zero net charge.
  • the molecule stops migrating at its pI because it is no longer affected by the electrical current.
  • the focusing chamber of the Rotofor is separated into twenty smaller chambers by permeable membranes. These twenty samples are removed simultaneously to ensure as little mixing as possible.
  • a sample is placed in the focusing medium, a buffered solution (see manufacturer's instructions) which includes 12.5% (w/v) glycerol and 2.5% of pH 3-10 Ampholytes (Bio-Rad).
  • a buffered solution see manufacturer's instructions
  • the fractions are collected, the pH of each determined and each fraction dialyzed against 1 M NaCl using a 3,500 MWCO membrane to remove the Ampholytes.
  • the samples are then dialyzed against deionized water to remove the NaCl.
  • Each fraction is lyophilized and resuspended in 0.4 ml of 10 mM NaCl.
  • the Rotofor fractions containing active material can be determined by protein assay and bioassay with insect larvae. The Rotofor fractions can then be subjected to further treatment or separation as described above.
  • Bioassays were conducted using CRW neonate larvae reared on artificial diets containing Pentin-1 obtained from P. macroloba as described herein.
  • the Pentin-1 may be from crude extract or purified as taught herein.
  • Pentin-1 was either topically applied to the diet surface or incorporated into the diet as taught by Czapla and Lang, J. Econo. Ento. 83(6):2480-2485 (1990).
  • the culture tray used in the bioassays were divided into treatment groups.
  • One or a plurality of Pentin-1 preparations or fractions from the various separations were screened in each tray; each preparation or fraction being applied to a plurality of cells. Each cell was infested with one or two neonate larvae.
  • a Mylar film with ventilation holes was affixed to the top of each tray to prevent escape and allow air exchange.
  • PBS phosphate buffered saline
  • Stoneville medium was prepared in standard fashion, but with only 90% of the prescribed water. Pentin-1 was added such that the amount in the diet was in the range of 1-5 ⁇ g/g.
  • the control treatment consisted of 0.9 ml PBS buffer added to 8.1 g of medium. The medium was poured into the cells and the cells were then infested and covered as described above. Insect weights (Weight or Avg. Wt.) were determined at Day 7 and are given in the tables.
  • Pentin-1 was purified and its insecticidal activity was established, cloning efforts were undertaken.
  • the first step of the process was to determine by western blot analysis the temporal and spatial distribution of Pentin-1 in order to identify the plant or seed tissue or tissues most likely expressing this protein. Since Pentin-1 was not isolated in quantities which allowed for the production of antibodies, the protein was sequenced in order to permit the design of peptides for synthesis.
  • the amino acid sequence data for Pentin-1 is shown below in FIG. 1. Carboxy-terminus and the internal sequence of approximately forty percent of the Pentin-1 peptides was compiled from fifteen peptides purified from LysC and CNBr digestion of purified Pentin-1 protein. The NH2-terminal sequence was not identified during this process.
  • Antibodies were raised against five of the peptides.
  • the synthetic peptides used to produce antibodies are listed below.
  • Genomic DNA was isolated, codon-degenerate oligonucleotides based on peptides were used to PCR amplify genomic fragments. Exon sequence of the resulting clones was used to do RT-PCR with specific oligos, then RT-PCR experiments were performed to obtain at least a partial Pentin-1 cDNA for probing the expression library. Information obtained from the sequencing of random cDNA clones from a P. macroloba immature seed library was used to generate a nascent codon usage table. The data obtained indicated the P. macroloba tree has no strong codon usage bias and that the GC content is moderate. A matrix of degenerate forward and reverse primers corresponding to Pentin-1 peptides were selected for use.
  • the forward primer sequence was VVKRLAGYFDV (Pentin-1 amino acid Nos. 76-86: Val Val Lys Arg Leu Ala Gly Tyr Phe Asp Val) (SEQ ID NO:10) and the reverse primer sequence was ENMENLEK, (Pentin-1 amino acid Nos. 372-379: Glu Asn Met Glu Asn Leu Glu Lys) (SEQ ID NO:I 1). Due to the small amount of tissue available, the initial primer testing was conducted using genomic DNA derived from P. macroloba leaves. One of the sixty-four possible primer combinations yielded a 3.0 kb fragment which encoded the Pentin-1 peptide sequences.
  • the forward and reverse primer pair were then used to amplify a 0.8 kb cDNA fragment from the total RNA isolated from mature (30-40 mm) seeds. Subsequent screening of the mature seed expression library with this 0.8 kb cDNA probe produced several related clones, one of which is a 1.4 kb clone that encodes twelve of the fifteen peptide sequences from Pentin-1 (SEQ ID NO:1).
  • the nucleic acid sequence of the Pentin-1 clone was determined by standard procedures known to those skilled in the art.
  • the cDNA sequence and the predicted Pentin-1 protein sequence is provided in FIG. 1 and SEQ ID NO:1.
  • Bioassays were conducted against Western corn rootworm (WCR) utilizing sonicated E. coli that had been transformed with one of several plasmids listed (Table 1). Transformed cells were grown in approximately 25-35 ml of TB broth. The cells were harvested after 24 h by centrifugation. The pellet was resuspended in approximately 1 ml of PBS buffer and sonicated. The resulting mixture was then top loaded onto the surface of diet and then infested with neonate WCR larvae. Mortality was recorded after four days. A positive result indicated 100% mortality. A negative result indicated mortality less than 10%.
  • a similar experiment involved the use of transformed cells grown on an agar plate. The cells were scraped off after sufficient growth, suspended in a small amount of PBS buffer and then the solution was incorporated into the insect diet. A 4-day bioassay was also conducted with mortality recorded.
  • Table 1 displays the results of two replicated bioassays. All cells transformed with putative negative (non-lethal WCR genes) plasmids did not cause any larval mortality in either test. These plasmids are P7725, P88126, and P11426. The two plasmids that contain the coding sequence for Pentin-1, but not any promoters to produce the actual protein, PGEM and P11394 did not display any WCR activity. However, all plasmids containing the coding region for Pentin 1 (SEQ ID NO:1) and a functional expression cassette displayed excellent activity against WCR larvae. All such treatments had 100% mortality. Preliminary western blot analysis indicated that a protein similar in size to Pentin-1 was present in these cell extracts, but not in negative control samples. Activity was seen in both types of cell preparation and bioassays.
  • Cell digestion Cells were digested in enzyme solution at 27° C. for 3-5 hours with 50-60 RPM shaking speed. The cell wall was digested with cellulase and pectolyase to release the protoplasts.
  • the protoplast pellet was resuspended in 20 ml or 40 ml KMC solution.
  • the protoplast density and total protoplast yield was determined by counting the number of protoplasts with a hemacytometer.
  • the suspension was centrifuged to pellet the protoplasts.
  • the protoplast pellet was suspended in MaMg transformation solution in a concentration of 2 million protoplasts per ml.
  • the protoplasts were transferred into a 15 ml tube using plastic squeeze Pasteur pipette and centrifuged 8 min at 500 RPM to pellet the protoplasts.
  • Enzyme solution for digesting suspension cell Enzyme solution
  • Enzyme solution contains 3% cellulase RS and 0.3% pectolyase Y23 in protoplast solution.
  • Immature maize embryos from green house donor plants are bombarded with a plasmid containing the three Pentin-1 constructs plus a plasmid containing the selectable marker gene, PAT, (Wohlleben, W., Arnold, W., Broer, I., Hillemann, D., Strauch, E. and Puehler, A. "Nucleotide sequence of the phosphinothricin N-acetyltransferase gene from Streptomyces viridochromogenes Tue494 and its expression in Nicotiana tabacum" Gene 70:25-37 (1988) that confers resistance to the herbicide Bialophos by the following method:
  • Preparation of target tissue The ears are surface sterilized in 30% Chlorox bleach plus 0.5% Micro detergent for 20 minutes, and rinsed two times with sterile water. The immature embryos are excised and placed embryo axis side down (scutellum side up), 25 embryos per plate. These are cultured on 560L medium 4 days prior to bombardment, in the dark. The day of bombardment, the embryos are transferred to 560Y medium for 4 hours, arranged within the 2.5 cm target zone.
  • Each reagent is added sequentially to the tungsten particle suspension, while maintained on the multi-tube vortexer.
  • the plasmids are adjusted for a final 1:1 ratio by size.
  • the final mixture is sonicated briefly, and allowed to incubate under constant vortexing for ten minutes.
  • the tubes are centrifuged briefly, liquid removed, washed with 500 ml 100% ethanol, and centrifuged 30 seconds. Again the liquid is removed, and 105 ⁇ l 100% ethanol added to the final tungsten particle pellet.
  • the tungsten/DNA particles are briefly sonicated and 10 ⁇ l spotted onto the center of each macro-carrier and allowed to dry about 2 minutes before bombardment.
  • Particle Gun Treatment The sample plates are bombarded at level #4 in particle gun #HE34-1 or #HE34-2. All samples receive a single shot at 650 PSI, with a total of ten aliquots taken from each tube of prepared particles/DNA.
  • Plants are then transferred to inserts in flats (equivalent to 2.5" pot) containing potting soil and grown for 1 week in a growth chamber, subsequently grown an additional 1-2 weeks in the greenhouse, then transferred to classic 600 pots (1.6 gallon) and grown to maturity.
  • ## Dissolve 1.660 g of Calcium Chloride Dihydrate in 950.000 ml of polished D-I H 2 O. Then dissolve 4.629 of Ammnonium Sulfate; 4.000 g of Potassium Phosphate Monobasic KH2PO4; 1.850 g of Magnesium Sulfate 7-H 2 O, MgSO 4 , 7H2O; and 28.300 g of Potassium Nitrate into sequence. Bring up to volume with polished D-I H 2 O.
  • ## Dissolve 0.100 g of Nicotinic Acid; 0.020 g of Thiamine.HCL; 0.100 g of Pyridoxine.HCL; and 0.400 g of Glycine in 875.00 ml of polished D-I H2O in sequence. Bring up to volume with polished D-I H 2 O. Make in 400 ml portions. Thiamine.HCL & Pyridoxine.HCL are in Dark Descicator. Store for one month, unless contamination or precipitation occur, then make fresh stock.
  • ## Dissolve 0.100 g of Nicotinic Acid; 0.020 g of Thiamine.HCL; 0.100 g of Pyridoxine.HCL; and 0.400 g of Glycine in 875.00 ml of polished D-I H 2 O in sequence. Bring up to volume with polished D-I H 2 O. Make in 400 ml portions. Thiamine.HCL & Pyridoxine.HCL are in Dark Descicator. Store for one month, unless contamination or precipitation occur, then make fresh stock.
  • PHP 11361 and PHP 11511 were deposited with the American Type Culture Collection, Bethesda, Md., and given Accession Nos. 209026 and 209025, respectively.
  • PHP11361 comprises the nucleotide sequence of the native Pentin-1 sequence.
  • PHP11511 comprises the optimized Pentin-1 sequence.

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  • Environmental Sciences (AREA)
  • Agricultural Chemicals And Associated Chemicals (AREA)
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US09/074,912 US6057491A (en) 1997-05-29 1998-05-08 Protein having insecticidal activities and method of use
BR9809516-1A BR9809516A (pt) 1997-05-29 1998-05-15 Proteìnas com atividades inseticidas e método de uso
CZ0421199A CZ297319B6 (cs) 1997-05-29 1998-05-15 Polypeptidy s insekticidní úcinností a jejich pouzití
SK1600-99A SK160099A3 (en) 1997-05-29 1998-05-15 Proteins having insecticidal activities and method of use
EP98921234A EP0986645A1 (en) 1997-05-29 1998-05-15 Proteins having insecticidal activities and method of use
CA002290776A CA2290776C (en) 1997-05-29 1998-05-15 Proteins having insecticidal activities and method of use
PCT/US1998/009995 WO1998054327A1 (en) 1997-05-29 1998-05-15 Proteins having insecticidal activities and method of use
AU73891/98A AU742709B2 (en) 1997-05-29 1998-05-15 Proteins having insecticidal activities and method of use
HU0003403A HUP0003403A3 (en) 1998-05-08 1998-05-15 Proteins having insecticidal activities and method of use
SI9820048A SI20214B (en) 1997-05-29 1998-05-15 Proteins having insecticidal activities and method of use
NZ501218A NZ501218A (en) 1997-05-29 1998-05-15 Proteins isolated from plant genus Pentaclethra that have insecticidal activities and method of use
CA002358161A CA2358161A1 (en) 1997-05-29 1998-05-15 Organisms containing insecticidal proteins
ARP980102483A AR016059A1 (es) 1997-05-29 1998-05-28 Proteina sustancialmente purificada que presenta propiedades insecticidas, secuencias de adn, vector, molecula aislada de nucleotidos, organismo, metodopara controlar el gusano de la raiz del maiz, celula vegetal transformada.
US09/290,136 US6339144B1 (en) 1997-05-29 1999-04-13 Proteins having insecticidal activities and method of use

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US6500617B1 (en) 1998-05-01 2002-12-31 Maxygen, Inc. Optimization of pest resistance genes using DNA shuffling
US20040091922A1 (en) * 2000-05-23 2004-05-13 Russell William Michael Lactobacillus beta-glucuronidase and DNA encoding the same
US20050188439A1 (en) * 2004-02-20 2005-08-25 Pioneer Hi-Bred International, Inc. Methods for enhancing insect resistance in plants
WO2006028495A2 (en) 2004-02-20 2006-03-16 Pioneer Hi-Bred International, Inc. Lipases and methods of use
US20060063243A1 (en) * 1989-09-27 2006-03-23 Van Gorcom Robert F Cloning and expression of microbial phytase
US7285404B1 (en) * 1999-09-07 2007-10-23 Meiji Seika Kaisha, Ltd. Cyclic depsipeptide synthetase and method for recombinant production
US20080293087A1 (en) * 2006-10-31 2008-11-27 Makielski Jonathan C Sulfonylurea receptor short forms from mitochondria and uses thereof
US20090215132A1 (en) * 2001-08-28 2009-08-27 Osamu Katoh Novel amide hydrolase gene
US10743535B2 (en) 2017-08-18 2020-08-18 H&K Solutions Llc Insecticide for flight-capable pests

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US6657046B1 (en) 2000-01-06 2003-12-02 Monsanto Technology Llc Insect inhibitory lipid acyl hydrolases
US6643672B1 (en) * 2000-07-31 2003-11-04 Hewlett-Packard Development Company, Lp. Method and apparatus for asynchronous file writes in a distributed file system
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MX339784B (es) 2011-03-30 2016-06-09 Univ Nac Autónoma De México Genes cry de bacillus thuringiensis mutantes y metodos de uso.
BR112013028318A2 (pt) 2011-05-02 2017-01-10 Pioneer Hi Bred Int método para identificar pelo menos um polinucleotídeo que codifica um polipeptídeo que tem atividade pesticida
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Cited By (12)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20060063243A1 (en) * 1989-09-27 2006-03-23 Van Gorcom Robert F Cloning and expression of microbial phytase
US6500617B1 (en) 1998-05-01 2002-12-31 Maxygen, Inc. Optimization of pest resistance genes using DNA shuffling
US7285404B1 (en) * 1999-09-07 2007-10-23 Meiji Seika Kaisha, Ltd. Cyclic depsipeptide synthetase and method for recombinant production
US20040091922A1 (en) * 2000-05-23 2004-05-13 Russell William Michael Lactobacillus beta-glucuronidase and DNA encoding the same
US20090215132A1 (en) * 2001-08-28 2009-08-27 Osamu Katoh Novel amide hydrolase gene
US7977073B2 (en) * 2001-08-28 2011-07-12 Mitsubishi Rayon Co., Ltd. Polynucleotide encoding a thermostable amide hydrolase and methods for producing an L-α-amino acid
US20050188439A1 (en) * 2004-02-20 2005-08-25 Pioneer Hi-Bred International, Inc. Methods for enhancing insect resistance in plants
WO2006028495A2 (en) 2004-02-20 2006-03-16 Pioneer Hi-Bred International, Inc. Lipases and methods of use
WO2006028495A3 (en) * 2004-02-20 2006-06-08 Pioneer Hi Bred Int Lipases and methods of use
US20060212964A9 (en) * 2004-02-20 2006-09-21 Pioneer Hi-Bred International, Inc. Methods for enhancing insect resistance in plants
US20080293087A1 (en) * 2006-10-31 2008-11-27 Makielski Jonathan C Sulfonylurea receptor short forms from mitochondria and uses thereof
US10743535B2 (en) 2017-08-18 2020-08-18 H&K Solutions Llc Insecticide for flight-capable pests

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